Asparaginase enzyme variants and uses thereof

ABSTRACT

The present invention relates to newly identified asparaginase polypeptide variants of SEQ ID NO: 3 and to polynucleotide sequences that encode such novel asparaginase variants. Furthermore the invention relates to the use of these novel asparaginase variants in industrial processes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation application of U.S. application Ser.No. 12/596,710, filed Oct. 20, 2009, which is a §371 National StageApplication of PCT/EP2008/054692, filed Apr. 17, 2008, which claimspriority to European Application No. 07106660.9, filed Apr. 20, 2007,European Application No. 0716662.5, filed Apr. 20, 2007, EuropeanApplication No. 07106620.3, filed Apr. 20, 2007, European ApplicationNo. 07106612.0, filed Apr. 20, 2007, and European Application No.07106664.1, filed Apr. 20, 2007, the content of all of which are hereinincorporated by reference in their entireties.

BACKGROUND

1. Field of the Invention

The invention relates to asparaginase polypeptide variants and topolynucleotide sequences comprising genes that encode these asparaginasevariants. The invention features a method for identifying suitableasparaginase variants. The invention also relates to methods of usingthese variant proteins in industrial processes. Also included in theinvention are cells transformed with a polynucleotide according to theinvention suitable for producing these proteins and cells, wherein aprotein according to the invention is genetically modified to enhance orreduce its activity and/or level of expression. The invention alsorelates to methods of using the asparaginase variants in industrialprocesses.

2. Description of Related Art

Recently, the occurrence of acrylamide in a number of heated foodproducts was published (Tareke et al. Chem. Res. Toxicol. 13, 517-522(2000)). Since acrylamide is considered as probably carcinogenic foranimals and humans, this finding had resulted in world-wide concern.Further research revealed that considerable amounts of acrylamide aredetectable in a variety of baked, fried and oven prepared common foodsand it was demonstrated that the occurrence of acrylamide in food wasthe result of the heating process.

A pathway for the formation of acrylamide from amino acids and reducingsugars as a result of the Maillard reaction has been proposed by Mottramet al. Nature 419:448 (2002). According to this hypothesis, acrylamidemay be formed during the Maillard reaction. During baking and roasting,the Maillard reaction is mainly responsible for the color, smell andtaste. A reaction associated with the Maillard is the Streckerdegradation of amino acids and a pathway to acrylamide was proposed. Theformation of acrylamide became detectable when the temperature exceeded120° C., and the highest formation rate was observed at around 170° C.When asparagine and glucose were present, the highest levels ofacrylamide could be observed, while glutamine and aspartic acid onlyresulted in trace quantities.

The official migration limit in the EU for acrylamide migrating intofood from food contact plastics is set at 10 ppb (10 micrograms perkilogram). Although no official limit is yet set for acrylamide thatforms during cooking, the fact that a lot of products exceed this value,especially cereals, bread products and potato or corn based products,causes concern.

Several plant raw materials are known to contain substantial levels ofasparagine. In potatoes asparagine is the dominant free amino acid (940mg/kg, corresponding with 40% of the total amino-acid content) and inwheat flour asparagine is present as a level of about 167 mg/kg,corresponding with 14% of the total free amino acids pool (Belitz andGrosch in Food Chemistry—Springer New York, 1999). The fact thatacrylamide is formed mainly from asparagine (combined with reducingsugars) may explain the high levels acrylamide in fried, oven-cooked orroasted plant products. Therefore, in the interest of public health,there is an urgent need for food products that have substantially lowerlevels of acrylamide or, preferably, are devoid of it.

A variety of solutions to decrease the acrylamide content has beenproposed, either by altering processing variables, e.g. temperature orduration of the heating step, or by chemically or enzymaticallypreventing the formation of acrylamide or by removing formed acrylamide.

In several patent applications the use of asparaginase for decreasingthe level of asparagine and thereby the amount of acrylamide formed hasbeen disclosed. Suitable asparaginases for this purpose have beenyielded from several fungal sources, as for example Aspergillus niger inWO2004/030468 and Aspergillus oryzae in WO04/032648.

Although all L-asparaginases catalyze the same chemical conversion, thisdoes not mean that they are suitable for the same applications. Variousapplications will place different demands on the conditions under whichthe enzymes have to operate. Physical and chemical parameters that mayinfluence the rate of an enzymatic conversion are the temperature (whichhas a positive effect on the chemical reaction rates, but may have anegative effect on enzyme stability), the moisture content, the pH, thesalt concentration, the structural integrity of the food matrix, thepresence of activators or inhibitors of the enzyme, the concentration ofthe substrate and products, etc.

Therefore there exists an ongoing need for improved asparaginases forseveral applications having improved properties.

OBJECT OF THE INVENTION

It is an object of the invention to provide novel asparaginase variantpolypeptides and polynucleotides encoding such variants. A furtherobject is to provide recombinant strains producing such asparaginasevariants. Also, a method for identifying variants is part of theinvention, as well as methods of making and using the polynucleotidesand polypeptides according to the invention.

SUMMARY OF THE INVENTION

The invention provides a polypeptide variant of a parent polypeptidehaving asparaginase activity, wherein the variant has an amino acidsequence which, when aligned with the sequence set out in SEQ ID NO: 3,comprises a substitution of an amino acid residue corresponding to anyof amino acids

41, 53, 62, 63, 64, 66, 70, 71, 73, 74, 76, 77, 88, 90, 91, 101, 102,103, 104, 106, 107, 108, 109, 111, 119, 122, 123, 132, 140, 142, 143,145, 161, 162, 163, 164, 168, 169, 170, 195, 211, 213, 214, 215, 216,217, 218, 219, 220, 228, 232, 233, 234, 235, 262, 267, 268, 269, 270,271, 272, 273, 293, 295, 297, 298, 299, 300, 301, 302, 303, 304, 310,314, 317, 318, 319, 321, 323, 324, 325, 327, 328, 329, 330, 331, 332,333, 334, 335 or 371said positions being defined with reference to SEQ ID NO: 3.

The invention also provides:

a nucleic acid sequence encoding such a variant;

a nucleic acid construct comprising a nucleic acid sequence encoding avariant of the invention operably linked to one or more controlsequences capable of directing the expression of an asparaginase in asuitable expression host;

a recombinant expression vector comprising such a nucleic acidconstruct;

a recombinant host cell comprising such an expression vector;

a method for producing an asparaginase comprising cultivating such ahost cell under conditions conducive to production of the asparaginaseand recovering the asparaginase;

a method of producing an asparaginase polypeptide variant, which methodcomprises:

-   a) selecting a parent asparaginase polypeptide;-   b) substituting at least one amino acid residue corresponding to any    of    -   41, 53, 62, 63, 64, 66, 70, 71, 73, 74, 76, 77, 88, 90, 91, 101,        102, 103, 104, 106, 107, 108, 109, 111, 119, 122, 123, 132, 140,        142, 143, 145, 161, 162, 163, 164, 168, 169, 170, 195, 211, 213,        214, 215, 216, 217, 218, 219, 220, 228, 232, 233, 234, 235, 262,        267, 268, 269, 270, 271, 272, 273, 293, 295, 297, 298, 299, 300,        301, 302, 303, 304, 310, 314, 317, 318, 319, 321, 323, 324, 325,        327, 328, 329, 330, 331, 332, 333, 334, 335 or 371

said positions being defined with reference to SEQ ID NO: 3.

-   c) optionally substituting one or more further amino acids as    defined in b);-   d) preparing the variant resulting from steps a)-c);-   e) determining the specific activity and/or the pH optimum of the    variant; and-   f) selecting a variant having an increased specific activity and/or    pH optimum in comparison to the parent asparaginase polypeptide,    thereby to produce an asparaginase polypeptide variant.

a composition comprising a variant of the invention or a variantobtained by a method of the invention;

use of a composition of the invention in the production of a foodproduct; and

use of a composition of the invention to reduce the amount of acrylamideformed in a thermally processed food product based on anasparagine-containing raw material.

DETAILED DESCRIPTION OF THE INVENTION

Throughout the present specification and accompanying claims, the words“comprise” and “include” and variations such as “comprises”,“comprising”, “includes” and “including” are to be interpretedinclusively. That is, these words are intended to convey the possibleinclusion of other elements or integers not specifically recited, wherethe context allows.

The present invention relates to a polypeptide variant of a parentpolypeptide having asparaginase activity. The variant has an amino acidsequence which, when aligned with the sequence set out in SEQ ID NO: 3,comprises a substitution of an amino acid residue corresponding to anyof amino acids

41, 53, 62, 63, 64, 66, 70, 71, 73, 74, 76, 77, 88, 90, 91, 101, 102,103, 104, 106, 107, 108, 109, 111, 119, 122, 123, 132, 140, 142, 143,145, 161, 162, 163, 164, 168, 169, 170, 195, 211, 213, 214, 215, 216,217, 218, 219, 220, 228, 232, 233, 234, 235, 262, 267, 268, 269, 270,271, 272, 273, 293, 295, 297, 298, 299, 300, 301, 302, 303, 304, 310,314, 317, 318, 319, 321, 323, 324, 325, 327, 328, 329, 330, 331, 332,333, 334, 335 or 371said positions being defined with reference to SEQ ID NO: 3.

That is to say, when the variant asparaginase sequence is aligned withthe sequence of the asparaginase of SEQ ID NO: 3, the variant willcomprise at least one substitution at a position (in the variant)corresponding to one of the positions set out above in SEQ ID NO: 3. A“substitution” in this context indicates that a position in the variantwhich corresponds to one of the positions set out above in SEQ ID NO: 3comprises an amino acid residue which does not appear at that positionin the parent polypeptide (which parent polypeptide may be SEQ ID NO:3).

Those positions in a variant asparaginase polypeptide of the inventionwhich correspond to the positions set out above in SEQ ID NO: 3 may beidentified by aligning the sequence of the variant polypeptide with thatof SEQ ID NO: 3 using, for example, the GAP alignment to the mosthomologous sequence found by the GAP program (see below for details ofthis program). The positions in the variant corresponding to thepositions in SEQ ID NO: 3 as set out above may thus be identified andare referred to as those positions defined with reference to SEQ ID NO:3.

The parent asparaginase polypeptide that may be used in the presentinvention may be any asparaginase (EC 3.5.1.1). A suitable asparaginasepolypeptide may be obtained from various sources, such as for examplefrom a plant, an animal or a microorganism. For example, an asparaginasemay be obtained from Escherichia, Erwinia, Streptomyces, Pseudomonas,Aspergillus and Bacillus species. An example of a suitable Escherichiastrain is Escherichia coli. An example of a suitable Erwinia strain isErwinia chrysanthemi. Examples of suitable Streptomyces strains areStreptomyces lividans and Streptomyces murinus. Examples of suitableAspergillus strains are Aspergillus oryzae, Aspergillus nidulans orAspergillus niger. Examples of suitable Bacillus strains are Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacilluscirculans, Bacillus coagulans, Bacillus lautus, Bacillus lentus,Bacillus licheniformis, Bacillus megaterium, Bacillusstearothermophilus, Bacillus subtilis or Bacillus thurigiensis.

An example of methods suitable for obtaining asparaginase from Bacillus,Streptomyces, Escherichia or Pseudomonas strains is described in WO03/083043. An example of methods suitable for obtaining asparaginasefrom Aspergillus is described in WO 2004/030468 and WO 04/032468.

A preferred parent asparaginase polypeptide suitable for use in theinvention is the polypeptide having the sequence set out in SEQ ID NO: 3or having at least 80% homology with SEQ ID NO: 3, for example at least85% homology with SEQ ID NO: 3, such as a least 85% homology with SEQ IDNO: 3, such as at least 90% homology with SEQ ID NO: 3, for example atleast 95%, at least 98% or at least 99% homology with SEQ ID NO: 3.

The amino acid residues in a variant of the invention that aresubstituted with comparison with the sequence set out in SEQ ID NO: 3are those which correspond to positions

41, 53, 62, 63, 64, 66, 70, 71, 73, 74, 76, 77, 88, 90, 91, 101, 102,103, 104, 106, 107, 108, 109, 111, 119, 122, 123, 132, 140, 142, 143,145, 161, 162, 163, 164, 168, 169, 170, 195, 211, 213, 214, 215, 216,217, 218, 219, 220, 228, 232, 233, 234, 235, 262, 267, 268, 269, 270,271, 272, 273, 293, 295, 297, 298, 299, 300, 301, 302, 303, 304, 310,314, 317, 318, 319, 321, 323, 324, 325, 327, 328, 329, 330, 331, 332,333, 334, 335 or 371as defined in relation to the sequence of SEQ ID NO: 3.

A variant may comprises a substitution at one or more of the saidpositions, for example at two, three, four, at least 5, at least 10, atleast 15, at least 20, at least 25, at least 30, at least 35, at least40, at least 45, at least 50, at least 55, at least 60, at least 65, atleast 70 or at all of the said positions.

A preferred subset of positions for substitution is defined by those atpositions

41, 53, 62, 63, 64, 66, 70, 71, 73, 74, 76, 77, 88, 90, 91, 101, 102,103, 104, 106, 107, 108, 109, 111, 119, 122, 123, 132, 140, 142, 143,145, 161, 162, 163, 164, 168, 169, 170, 195, 211, 214, 215, 216, 218,220, 228, 232, 233, 262, 267, 293, 295, 297, 298, 299, 300, 301, 303,304, 310, 314, 317, 319, 321, 324, 330, 332, 333, 334 or 371.as defined in relation to the sequence of SEQ ID NO: 3.

A variant may comprises a substitution at one or more of the saidpositions, for example at two, three, four, at least 5, at least 10, atleast 15, at least 20, at least 25, at least 30, at least 35, at least40, at least 45 or at all of the said positions.

A more preferred subset of positions for substitution is defined bythose at positions

41, 53, 63, 64, 66, 73, 74, 76, 77, 88, 90, 91, 101, 106, 111, 119, 122,123, 132, 140, 145, 161, 170, 195, 211, 218, 228, 232, 233, 262, 267,293, 295, 297, 299, 300, 301, 303, 304, 310, 314, 317, 321, 324, 330,332, 333 or 371,as defined in relation to the sequence of SEQ ID NO: 3.A even more preferred subset of positions for substitution is defined bythose at positions53, 63, 64, 66, 73, 74, 76, 77, 88, 101, 140, 170, 293,as defined in relation to the sequence of SEQ ID NO: 3.

A variant may comprises a substitution at one or more of the saidpositions, for example at two, three, four, at least 5, at least 10, atleast 15, at least 20 or at all of the said positions.

A variant of the invention comprises one or more substitutions asdefined above. A “substitution” in this context indicates that aposition in the variant which corresponds to one of the positions setout above in SEQ ID NO: 3 comprises an amino acid residue which does notappear at that position in the parent polypeptide (the parent may be SEQID NO: 3).

Preferred substitutions are set out in the following Table (with thepositions being defined in relation to the sequence set out in SEQ IDNO: 3).

Most More Position preferred preferred Preferred  41 I IN  53 Y LY  62GAT GATFK  63 GVS GASV GASIVE  64 P ANDP  66 P NKP  70 AS  71 N ASNE  73K QNKE HSNDQERK  74 A AV  76 T STV STVQNKE  77 I IL  88 YP YPE  90 V VYF 91 E SNE 101 V VSTDH 102 S SRK 103 I LIFMT 104 ND AND 106 P PQGAKESTNPQ 107 N SNE 108 VM VML 109 NS RDGNS 111 G GS GSTH 119 N TNTNMQER 122 H EH ADEHK 123 A ALT 132 S 140 N 142 M 143 D DSAG 145 S S 161L VL VLFM 162 T AT 163 A 164 S GS 168 A AGT 169 S AGS 170 T ST EGST 195D 211 S SV SVMINQ 213 S SIM 214 H SH 215 ST 216 ST STVLF 217 SN ASNDK218 V VLT 219 NQE ASNQE 220 AS 228 H NH ASNH 232 v VI VIF 233 V VHVHLREYFS 234 ND GND 235 GS GSDI 262 CH 267 Y Y 268 NAG GANTF 269 HF 270Q ASIQ 271 N NE 272 A AIDQ 273 QTS QTSPDE 293 SV SVET SVETML 295 S NS297 S NSTA 298 ILM ILMWFT 299 S SDA SDAPHYN 300 I SI EDHKANQIS 301 PTPAGD GATPNRDEYK 302 Y QNHWVIY 303 S YFS GLKEDIAYFS 304 T ATSD ATSDNKP310 V ATV AVMT 314 D SND GASNDQH 317 I I 318 MIA 319 ATLR ATLRVIYH 321 TST STHRKA 323 VS IVS 324 G GP MAGP 325 AST ASTDEW 327 M MIPYSARVT 328 STSTI 329 AT ATLG 330 S PSYTIL DERQVPSYTIL 331 AT ATGNDEKR 332 A AANSGEKPQ 333 E EDS EDSIFAGK 334 GTDE GTDEPVI 335 TGD 371 M AIM

A variant according to the invention may have an amino acid sequencewhich, when aligned with the sequence set out in SEQ ID NO: 3, comprisesone or more of

Ile at position 41, Tyr at position 53, Gly or Val or Ser at position63, Pro at position 64, Pro at position 66, Lys at position 73, Ala atposition 74, Thr at position 76, Ile at position 77, Tyr or Pro atposition 88, Val at position 90, Glu at position 91, Val at position101, Pro at position 106, Gly at position 111, Asn at position 119, Hisat position 122, Ala at position 123, Ser at position 132, Asn atposition 140, Ser at position 145, Leu at position 161, Thr at position170, Asp at position 195, Ser at position 211, Val at position 218, Hisat position 228, Val at position 232, Val at position 233, Cys or His atposition 262, Tyr at position 267, Ser or Val at position 293, Ser atposition 295, Ser at position 297, Ser at position 299, Ile at position300, Pro at position 301, Ser at position 303, Thr at position 304, Valat position 310, Asp at position 314, Ile at position 317, Thr atposition 321, Gly at position 324, Ser at position 330, Ala at position332, Glu at position 333 or Met at position 371said positions being defined with reference to SEQ ID NO: 3.

In a preferred embodiment a variant according to the invention may havean amino acid sequence which, when aligned with the sequence set out inSEQ ID NO: 3, comprises one or more of

Tyr at position 53, Gly or Val at position 63, Pro at position 64, Proat position 66, Lys at position 73, Ala at position 74, Thr at position76, Ile at position 77, Tyr or Pro at position 88, Val at position 101,Asn at position 140, Thr at position 170, Ser or Val at position 293,said positions being defined with reference to SEQ ID NO: 3.

In a preferred embodiment a variant according to the invention may havean amino acid sequence which, when aligned with the sequence set out inSEQ ID NO: 3, comprises at least one substitution of an amino acidresidue corresponding to amino acid 63, preferably comprising a Gly orVal at position 63, said positions being defined with reference to SEQID NO: 3 and wherein the parent polypeptide of said variant preferablycorresponds to SEQ ID NO: 3.

Examples of a variant of a parent polypeptide having asparaginaseactivity, wherein the variant has an amino acid sequence which, whenaligned with the sequence set out in SEQ ID NO: 3, comprises at leastone substitution of an amino acid residue corresponding to amino acid63, said positions being defined with reference to SEQ ID NO: 3. andwherein the parent polypeptide corresponds to SEQ ID NO: 3, are thepolypeptide comprising the substitutions D63G and G132S (tentativelycalled ASN01), the polypeptide comprising the substitutions D63G, D111Gand R122H (tentatively called ASN02), the polypeptide comprising thesubstitutions D63V and T3001 (tentatively called ASN03).

In another embodiment a variant according to the invention may have anamino acid sequence which, when aligned with the sequence set out in SEQID NO: 3, comprises at least one substitution of an amino acid residuecorresponding to amino acid 64, preferably comprising a Pro at position64,

said positions being defined with reference to SEQ ID NO: 3 and whereinthe parent polypeptide of said variant preferably corresponds to SEQ IDNO: 3.

Examples of a variant of a parent polypeptide having asparaginaseactivity, wherein the variant has an amino acid sequence which, whenaligned with the sequence set out in SEQ ID NO: 3, comprises at leastone substitution of an amino acid residue corresponding to amino acid64, said positions being defined with reference to SEQ ID NO: 3. andwherein the parent polypeptide corresponds to SEQ ID NO: 3, is thepolypeptide comprising the substitutions S64P and 1310V (tentativelycalled ASN04).

In yet another embodiment a variant according to the invention may havean amino acid sequence which, when aligned with the sequence set out inSEQ ID NO: 3, comprises at least one substitution of an amino acidresidue corresponding to amino acid 66, preferably comprising a Pro atposition 66, said positions being defined with reference to SEQ ID NO: 3and wherein the parent polypeptide of said variant preferablycorresponds to SEQ ID NO: 3.

Examples of a variant of a parent polypeptide having asparaginaseactivity, wherein the variant has an amino acid sequence which, whenaligned with the sequence set out in SEQ ID NO: 3, comprises at leastone substitution of an amino acid residue corresponding to amino acid66, said positions being defined with reference to SEQ ID NO: 3. andwherein the parent polypeptide corresponds to SEQ ID NO: 3, is thepolypeptide comprising the substitutions T411, S66P and V371M(tentatively called ASN05).

In yet another embodiment a variant according to the invention may havean amino acid sequence which, when aligned with the sequence set out inSEQ ID NO: 3, comprises at least one substitution of an amino acidresidue corresponding

to amino acid 73, 74, 293,preferably comprising one or more of Lys at position 73, Ala at position74, Ser or Val at position 293said positions being defined with reference to SEQ ID NO: 3 and whereinthe parent polypeptide of said variant preferably corresponds to SEQ IDNO: 3.

Examples of a variant of a parent polypeptide having asparaginaseactivity, wherein the variant has an amino acid sequence which, whenaligned with the sequence set out in SEQ ID NO: 3, comprises at leastone substitution of an amino acid residue corresponding to amino acid73, 74 or 293, said positions being defined with reference to SEQ ID NO:3. and wherein the parent polypeptide corresponds to SEQ ID NO: 3, arethe polypeptide comprising the substitutions G195D and A293V(tentatively called ASN14), the polypeptide comprising the substitutionsT73K; S74A; and A293S (tentatively called ASN15) or the polypeptidecomprising the substitutions T73K, S74A, E106P, A293S, G297S, T299S,Q319A, M321T, and V324G (tentatively called ASN16).

In a further embodiment a variant according to the invention may have anamino acid sequence which, when aligned with the sequence set out in SEQID NO: 3, comprises at least one substitution of an amino acid residuecorresponding

to amino acid 76 or 101preferably comprising one or more of Thr at position 76, or Val atposition 101said positions being defined with reference to SEQ ID NO: 3 and whereinthe parent polypeptide of said variant preferably corresponds to SEQ IDNO: 3.

Examples of a variant of a parent polypeptide having asparaginaseactivity, wherein the variant has an amino acid sequence which, whenaligned with the sequence set out in SEQ ID NO: 3, comprises at leastone substitution of an amino acid residue corresponding to amino acid 76or 101, said positions being defined with reference to SEQ ID NO: 3. andwherein the parent polypeptide corresponds to SEQ ID NO: 3, is thepolypeptide comprising the substitutions A76T and A101V (tentativelycalled ASN06).

In yet a further embodiment a variant according to the invention mayhave an amino acid sequence which, when aligned with the sequence setout in SEQ ID NO: 3, comprises at least one substitution of an aminoacid residue corresponding

to amino acid 77, preferably comprising a Ile at position 77said positions being defined with reference to SEQ ID NO: 3 and whereinthe parent polypeptide of said variant preferably corresponds to SEQ IDNO: 3.

Examples of a variant of a parent polypeptide having asparaginaseactivity, wherein the variant has an amino acid sequence which, whenaligned with the sequence set out in SEQ ID NO: 3, comprises at leastone substitution of an amino acid residue corresponding to amino acid77, said positions being defined with reference to SEQ ID NO: 3. andwherein the parent polypeptide corresponds to SEQ ID NO: 3, is thepolypeptide comprising the substitutions V771, V123A and E314D(tentatively called ASN07).

In an embodiment a variant according to the invention may have an aminoacid sequence which, when aligned with the sequence set out in SEQ IDNO: 3, comprises at least one substitution of an amino acid residuecorresponding to amino acid 88, preferably comprising a Tyr or Pro atposition 88

said positions being defined with reference to SEQ ID NO: 3 and whereinthe parent polypeptide of said variant preferably corresponds to SEQ IDNO: 3.

Examples of a variant of a parent polypeptide having asparaginaseactivity, wherein the variant has an amino acid sequence which, whenaligned with the sequence set out in SEQ ID NO: 3, comprises at leastone substitution of an amino acid residue corresponding to amino acid88, said positions being defined with reference to SEQ ID NO: 3. andwherein the parent polypeptide corresponds to SEQ ID NO: 3, is thepolypeptide comprising the substitution S88Y (tentatively called ASN08)or the polypeptide comprising the substitutions S88P, I161L and R262c(tentatively called ASN09).

In another embodiment a variant according to the invention may have anamino acid sequence which, when aligned with the sequence set out in SEQID NO: 3, comprises at least one substitution of an amino acid residuecorresponding to amino acid 140, preferably comprising a Asn at position140

said positions being defined with reference to SEQ ID NO: 3 and whereinthe parent polypeptide of said variant preferably corresponds to SEQ IDNO: 3.

Examples of a variant of a parent polypeptide having asparaginaseactivity, wherein the variant has an amino acid sequence which, whenaligned with the sequence set out in SEQ ID NO: 3, comprises at leastone substitution of an amino acid residue corresponding to amino acid140, said positions being defined with reference to SEQ ID NO: 3. andwherein the parent polypeptide corresponds to SEQ ID NO: 3, is thepolypeptide comprising the substitution D140N (tentatively calledASN10).

In one embodiment a variant according to the invention may have an aminoacid sequence which, when aligned with the sequence set out in SEQ IDNO: 3, comprises at least one substitution of an amino acid residuecorresponding to amino acid 170, preferably comprising a Thr at position170

said positions being defined with reference to SEQ ID NO: 3 and whereinthe parent polypeptide of said variant preferably corresponds to SEQ IDNO: 3.

Examples of a variant of a parent polypeptide having asparaginaseactivity, wherein the variant has an amino acid sequence which, whenaligned with the sequence set out in SEQ ID NO: 3, comprises at leastone substitution of an amino acid residue corresponding to amino acid170, said positions being defined with reference to SEQ ID NO: 3. andwherein the parent polypeptide corresponds to SEQ ID NO: 3, is thepolypeptide comprising the substitutions D91E, A170T and R262H(tentatively called ASN11).

In one more embodiment a variant according to the invention may have anamino acid sequence which, when aligned with the sequence set out in SEQID NO: 3, comprises at least one substitution of an amino acid residuecorresponding

to amino acid 53, preferably comprising a Tyr at position 53said positions being defined with reference to SEQ ID NO: 3 and whereinthe parent polypeptide of said variant preferably corresponds to SEQ IDNO: 3.

Examples of a variant of a parent polypeptide having asparaginaseactivity, wherein the variant has an amino acid sequence which, whenaligned with the sequence set out in SEQ ID NO: 3, comprises at leastone substitution of an amino acid residue corresponding to amino acid53, said positions being defined with reference to SEQ ID NO: 3. andwherein the parent polypeptide corresponds to SEQ ID NO: 3, is thepolypeptide comprising the substitutions F53Y and K119N (tentativelycalled ASN13).

A further examples of a variant of a parent polypeptide havingasparaginase activity, wherein the variant has an amino acid sequencewhich, when aligned with the sequence set out in SEQ ID NO: 3, comprisesat least one substitution of an amino acid residue corresponding to anyof amino acids

41, 53, 63, 64, 66, 73, 74, 76, 77, 88, 90, 91, 101, 106, 111, 119, 122,123, 132, 140, 145, 161, 170, 195, 211, 218, 228, 232, 233, 262, 267,293, 295, 297, 299, 300, 301, 303, 304, 310, 314, 317, 321, 324, 330,332, 333 or 371said positions being defined with reference to SEQ ID NO: 3.and wherein the parent polypeptide corresponds to SEQ ID NO: 3, is thepolypeptide comprising the substitutions L90V, K119N, Y228H, and R262c(tentatively called ASN12).

A variant of the invention may also comprise additional modifications incomparison to the parent at positions other than those specified above,for example, one or more additional substitutions, additions ordeletions. A variant of the invention may comprise a combination ofdifferent types of modification of this sort. A variant may compriseone, two, three, four, least 5, at least 10, at least 15, at least 20,at least 25, at least 30 or more such modifications (which may all be ofthe same type or may be different types of modification). Typically, theadditional modifications may be substitutions.

A variant according to the invention may have at least 80% homology withthe parent asparaginase polypeptide, for example at least 85% homologywith the parent polypeptide, such as 90% homology with the parentpolypeptide, at least 95% homology with the parent polypeptide, at least98% homology with the parent polypeptide or at least 99% homology withthe parent polypeptide.

A variant of the invention will typically retain asparaginase activity(EC 3.5.1.1). That is to say, a variant of the invention will typicallybe capable of catalysing the hydrolysis of asparagine to aspartic acid.A variant of the invention is, therefore, one which is typically capableof modifying the side chains of asparaginase that are involved in theformation of acrylamide during the production of a food productinvolving at least one heating step.

Preferably, a variant of the invention will typically exhibit improvedproperties in comparison with the parent asparaginase polypeptide fromwhich it is derived. Such an improved property will typically be onewhich is relevant if the variant were to be used as set out below, forexample in a method for preparing a foodstuff.

Such properties include, but are not limited to, increased specificactivity (such that it may be possible to use a smaller amount of thevariant in a method for the preparation of a foodstuff as compared tothe amount of parent asparaginase required), an increased or decreasedpH optimum, more particularly a pH optimum more suited for use in amethod for the preparation of a foodstuff (as compared to the parentasparaginase) and increased thermostability.

In one embodiment a variant protein according to the invention may havea pH optimum which is higher than that of the parent polypeptide orlower than the parent polypeptide. Preferably the pH optimum of thevariant protein is higher than that of the parent polypeptide.Preferably the parent polypeptide is that according to SEQ ID NO: 3. Forexample, the wild-type asparaginase from A. niger (as disclosed in SEQID NO: 3) has a pH optimum of from pH 4 to pH 5. A variant protein ofthe invention may be more alkaliphilic than such a wild-type enzyme,i.e. may, for example, have a pH optimum of from pH 5 to pH 8,preferably from pH 6 to pH 7. Optionally a variant protein of theinvention may be more acidophilic than the wild type asparaginase.

Preferably a variant asparaginase protein according to the invention mayhave a pH, which is higher than the pH optimum and at which 50% of theasparaginase activity is still present, (hereafter indicated as alkalinepH), which is higher than that of the parent asparaginase. When theparent asparaginase is that according to SEQ ID NO: 3 the variantprotein may have an alkaline pH at which 50% of the activity is observedwhich is at least 6.9, preferably, at least 7.0, at least 7.5,preferably at least 8.

A variant which exhibits a property which is improved in relation to theparent asparaginase is one which demonstrates a measurable reduction orincrease in the relevant property, typically such that the variant ismore suited to use as set out below, for example in a method for theproduction of a foodstuff.

Preferably a variant protein according to the invention may have aspecific activity which is higher than that of the parent polypeptidemeasured at the same pH. With specific activity of a variant protein itis herewith intended the asparaginase activity of the variant proteinmeasured in units/mg of pure protein. Preferably the specific actity ofthe variant protein according to the invention is higher at at least onepH, preferably a pH between 4 and 8, than that of the parent polypeptidemeasured at the same pH.

In another embodiment of the invention the variant asparaginase is morethermophilic than the parent asparaginase polypeptide.

The property may thus be decreased by at least 10%, at least 20%, atleast 30%, at least 40% at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95% or at least 99%. Alternatively,the property may be increased by at least 10%, at least 25%, at least50%, at least 100%, at least, 200%, at least 500% or at least 1000%. Thepercentage decrease or increase in this context represents thepercentage decrease or increase in comparison to the parent asparaginasepolypeptide. It is well known to the skilled person how such percentagechanges may be measured—it is a comparison of the activity of the parentasparaginase and the variant asparaginase.

The variants described herein are collectively comprised in the terms “apolypeptide according to the invention” or “a variant according to theinvention”.

The terms “peptide” and “oligopeptide” are considered synonymous (as iscommonly recognized) and each term can be used interchangeably as thecontext requires to indicate a chain of at least two amino acids coupledby peptidyl linkages. The word “polypeptide” is used herein for chainscontaining more than seven amino acid residues. All oligopeptide andpolypeptide formulas or sequences herein are written from left to rightand in the direction from amino terminus to carboxy terminus. Theone-letter code of amino acids used herein is commonly known in the artand can be found in Sambrook, et al. (Molecular Cloning: A LaboratoryManual, 2nd, ed. Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989).

By “isolated” polypeptide or protein is intended a polypeptide orprotein removed from its native environment. For example, recombinantlyproduced polypeptides and proteins expressed in host cells areconsidered isolated for the purpose of the invention as are recombinantpolypeptides which have been substantially purified by any suitabletechnique such as, for example, the single-step purification methoddisclosed in Smith and Johnson, Gene 67:31-40 (1988).

A polypeptide variant according to the invention can be recovered andpurified from recombinant cell cultures by methods known in the art.Most preferably, high performance liquid chromatography (“HPLC”) isemployed for purification.

Polypeptides of the present invention include products of chemicalsynthetic procedures, and products produced by recombinant techniquesfrom a prokaryotic or eukaryotic host, including, for example,bacterial, yeast, higher plant, insect and mammalian cells. Dependingupon the host employed in a recombinant production procedure, thepolypeptides of the present invention may be glycosylated or may benon-glycosylated. In addition, polypeptides of the invention may alsoinclude an initial modified methionine residue, in some cases as aresult of host-mediated processes.

The invention also features biologically active fragments of thepolypeptide variants according to the invention. Such fragments areconsidered to be encompassed within the term “a variant of theinvention”.

Biologically active fragments of a polypeptide variant of the inventioninclude polypeptides comprising amino acid sequences sufficientlyidentical to or derived from the amino acid sequence of a variantprotein of the invention which include fewer amino acids than the fulllength protein but which exhibit at least one biological activity of thecorresponding full-length protein. Typically, biologically activefragments comprise a domain or motif with at least one activity of avariant protein of the invention. A biologically active fragment of aprotein of the invention can be a polypeptide which is, for example, 10,25, 50, 100 or more amino acids in length. Moreover, other biologicallyactive portions, in which other regions of the protein are deleted, canbe prepared by recombinant techniques and evaluated for one or more ofthe biological activities of the native form of a polypeptide of theinvention.

Typically, a protein fragment of the invention will comprise one or moreof the substitutions defined herein.

The invention also features nucleic acid fragments which encode theabove biologically active fragments (which biologically active fragmentsare themselves variants of the invention).

As set out above, the present invention provides polynucleotidesencoding the variant polypeptides of the invention. The invention alsorelates to an isolated polynucleotide encoding at least one functionaldomain of a polypeptide variant of the invention. Typically, such adomain will comprise one or more of the substitutions described herein.

In one embodiment of the invention, the nucleic acid sequence accordingto the invention encodes a polypeptide, wherein the polypeptide is avariant comprising an amino acid sequence that has one or moretruncation(s), and/or at least one substitution, deletion and/orinsertion of an amino acid as compared to the parent asparaginase. Sucha polypeptide will, however, typically comprise one or more of thesubstitutions described herein.

As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules which include an open reading frame encoding a variant asdescribed herein. A gene may include coding sequences, non-codingsequences, introns and regulatory sequences. That is to say, a “gene”,as used herein, may refer to an isolated nucleic acid molecule asdefined herein. Accordingly, the term “gene”, in the context of thepresent application, does not refer only to naturally-occurringsequences.

A nucleic acid molecule of the present invention can be generated usingstandard molecular biology techniques well known to those skilled in theart taken in combination with the sequence information provided herein.

For example, using standard synthetic techniques, the required nucleicacid molecule may be synthesized de novo. Such a synthetic process willtypically be an automated process.

Alternatively, a nucleic acid molecule of the invention may be generatedby use of site-directed mutagenesis of an existing nucleic acidmolecule, for example a wild-type nucleic acid molecule. Site-directedmutagenesis may be carried out using a number of techniques well know tothose skilled in the art.

In one such method, mentioned here merely by way of example, PCR iscarried out on a plasmid template using oligonucleotide “primers”encoding the desired substitution. As the primers are the ends ofnewly-synthesized strands, should there be a mis-match during the firstcycle in binding the template DNA strand, after that first round, theprimer-based strand (containing the mutation) would be at equalconcentration to the original template. After successive cycles, itwould exponentially grow, and after 25, would outnumber the original,unmutated strand in the region of 8 million: 1, resulting in a nearlyhomogeneous solution of mutated amplified fragments. The template DNAmay then be eliminated by enzymatic digestion with, for example using arestriction enzyme which cleaves only methylated DNA, such as Dpn1. Thetemplate, which is derived from an alkaline lysis plasmid preparationand therefore is methylated, is destroyed in this step, but the mutatedplasmid is preserved because it was generated in vitro and isunmethylated as a result.

In such a method more than one mutation (encoding a substitution asdescribed herein) may be introduced into a nucleic acid molecule in asingle PCR reaction, for example by using one or more oligonucleotides,each comprising one or more mis-matches. Alternatively, more than onemutation may be introduced into a nucleic acid molecule by carrying outmore than one PCR reaction, each reaction introducing one or moremutations, so that altered nucleic acids are introduced into the nucleicacid in a sequential, iterative fashion.

A nucleic acid of the invention can be generated using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriate mis-matchedoligonucleotide primers according to the site-directed mutagenesistechnique described above. A nucleic acid molecule derived in this waycan be cloned into an appropriate vector and characterized by DNAsequence analysis.

A nucleic acid sequence of the invention may comprise one or moredeletions, i.e. gaps, in comparison to the parent asparaginase. Suchdeletions/gaps may also be generated using site-directed mutagenesisusing appropriate oligonucleotides. Techniques for generating suchdeletions are well known to those skilled in the art.

Furthermore, oligonucleotides corresponding to or hybridizable tonucleotide sequences according to the invention can be prepared bystandard synthetic techniques, e.g., using an automated DNA synthesizer.

Also, complementary nucleic acid molecules are included in the presentinvention. A nucleic acid molecule which is complementary to anothernucleotide sequence is one which is sufficiently complementary to theother nucleotide sequence such that it can hybridize to the othernucleotide sequence thereby forming a stable duplex.

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode a variant of the invention, or a biologically activefragment or domain thereof, as well as nucleic acid molecules sufficientfor use as hybridization probes to identify nucleic acid moleculesencoding a polypeptide of the invention and fragments of such nucleicacid molecules suitable for use as PCR primers for the amplification ormutation of nucleic acid molecules, such as for the preparation ofnucleic acid molecules of the invention.

An “isolated polynucleotide” or “isolated nucleic acid” is a DNA or RNAthat is not immediately contiguous with both of the coding sequenceswith which it is immediately contiguous (one on the 5′ end and one onthe 3′ end) in the naturally occurring genome of the organism from whichit is derived. Thus, in one embodiment, an isolated nucleic acidincludes some or all of the 5′ non-coding (e.g., promotor) sequencesthat are immediately contiguous to the coding sequence. The termtherefore includes, for example, a recombinant DNA that is incorporatedinto a vector, into an autonomously replicating plasmid or virus, orinto the genomic DNA of a prokaryote or eukaryote, or which exists as aseparate molecule (e.g., a cDNA or a genomic DNA fragment produced byPCR or restriction endonuclease treatment) independent of othersequences. It also includes a recombinant DNA that is part of a hybridgene encoding an additional polypeptide that is substantially free ofcellular material, viral material, or culture medium (when produced byrecombinant DNA techniques), or chemical precursors or other chemicals(when chemically synthesized). Moreover, an “isolated nucleic acidfragment” is a nucleic acid fragment that is not naturally occurring asa fragment and would not be found in the natural state.

As used herein, the terms “polynucleotide” or “nucleic acid molecule”are intended to include DNA molecules (e.g., cDNA or genomic DNA) andRNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated usingnucleotide analogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA. The nucleic acidmay be synthesized using oligonucleotide analogs or derivatives (e.g.,inosine or phosphorothioate nucleotides). Such oligonucleotides can beused, for example, to prepare nucleic acids that have alteredbase-pairing abilities or increased resistance to nucleases.

Another embodiment of the invention provides an isolated nucleic acidmolecule which is antisense to a nucleic acid molecule of the invention.

The terms “homology” or “percent identity” are used interchangeablyherein. For the purpose of this invention, it is defined here that inorder to determine the percent identity of two amino acid sequences ortwo nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in the sequence of afirst amino acid or nucleic acid for optimal alignment with a secondamino or nucleic acid sequence). The amino acid or nucleotide residuesat corresponding amino acid or nucleotide positions are then compared.When a position in the first sequence is occupied by the same amino acidor nucleotide residue as the corresponding position in the secondsequence, then the molecules are identical at that position. The percentidentity between the two sequences is a function of the number ofidentical positions shared by the sequences (i.e., % identity=number ofidentical positions/total number of positions (i.e. overlappingpositions)×100). Preferably, the two sequences are the same length.

A sequence comparison may be carried out over the entire lengths of thetwo sequences being compared or over fragment of the two sequences.Typically, the comparison will be carried out over the full length ofthe two sequences being compared. However, sequence identity may becarried out over a region of, for example, twenty, fifty, one hundred ormore contiguous amino acid residues.

The skilled person will be aware of the fact that several differentcomputer programs are available to determine the homology between twosequences. For instance, a comparison of sequences and determination ofpercent identity between two sequences can be accomplished using amathematical algorithm. In a preferred embodiment, the percent identitybetween two amino acid or nucleic acid sequences is determined using theNeedleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) algorithmwhich has been incorporated into the GAP program in the Accelrys GCGsoftware package (available at http://www.accelrys.com/products/gcg/),using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.The skilled person will appreciate that all these different parameterswill yield slightly different results but that the overall percentageidentity of two sequences is not significantly altered when usingdifferent algorithms.

The protein sequences or nucleic acid sequences of the present inventioncan further be used as a “query sequence” to perform a search againstpublic databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the BLASTN andBLASTP programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.215:403-10. BLAST protein searches can be performed with the BLASTPprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to protein molecules of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the defaultparameters of the respective programs (e.g., BLASTP and BLASTN) can beused. See the homepage of the National Center for BiotechnologyInformation at http://www.ncbi.nlm.nih.gov/.

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a a variantasparaginase of the invention.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments can beligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. The terms “plasmid” and “vector” can be usedinterchangeably herein as the plasmid is the most commonly used form ofvector. However, the invention is intended to include such other formsof expression vectors, such as viral vectors (e.g., replicationdefective retroviruses, adenoviruses and adeno-associated viruses),which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorincludes one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operatively linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signal). Such regulatory sequences aredescribed, for example, in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those which direct constitutive expression of anucleotide sequence in many types of host cells and those which directexpression of the nucleotide sequence only in a certain host cell (e.g.tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produceproteins or peptides, encoded by nucleic acids as described herein (e.g.the asparaginase variant of SEQ ID NO: 3 or a variant thereof, forexample a functional equivalent or fragment, or a fusion proteincomprising one or more of such variants).

The recombinant expression vectors of the invention can be designed forexpression of variant proteins of the invention in prokaryotic oreukaryotic cells. For example, a variant protein of the invention can beexpressed in bacterial cells such as E. coli, insect cells (usingbaculovirus expression vectors) yeast cells or mammalian cells. Suitablehost cells are discussed further in Goeddel, Gene Expression TechnologyMethods in Enzymology 185, Academic Press, San Diego, Calif. (1990).Alternatively, the recombinant expression vector can be transcribed andtranslated in vitro, for example using T7 promoter regulatory sequencesand T7 polymerase.

Expression vectors useful in the present invention include chromosomal-,episomal- and virus-derived vectors e.g., vectors derived from bacterialplasmids, bacteriophage, yeast episome, yeast chromosomal elements,viruses such as baculoviruses, papova viruses, vaccinia viruses,adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses,and vectors derived from combinations thereof, such as those derivedfrom plasmid and bacteriophage genetic elements, such as cosmids andphagemids.

The DNA insert should be operatively linked to an appropriate promoter,such as the phage lambda PL promoter, the E. coli lac, trp and tacpromoters, the SV40 early and late promoters and promoters of retroviralLTRs, to name a few. Other suitable promoters will be known to theskilled person. In a specific embodiment, promoters are preferred thatare capable of directing a high expression level of asparaginase infilamentous fungi. Such promoters are known in the art. The expressionconstructs may contain sites for transcription initiation, termination,and, in the transcribed region, a ribosome binding site for translation.The coding portion of the mature transcripts expressed by the constructswill include a translation initiating AUG at the beginning and atermination codon appropriately positioned at the end of the polypeptideto be translated.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-percipitation, DEAE-dextran-mediated transfection,transduction, infection, lipofection, cationic lipidmediatedtransfection or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MolecularCloning: A Laboratory Manual, 2nd, ed. Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),Davis et al., Basic Methods in Molecular Biology (1986) and otherlaboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methatrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding a variant protein of the invention or can beintroduced on a separate vector. Cells stably transfected with theintroduced nucleic acid can be identified by drug selection (e.g. cellsthat have incorporated the selectable marker gene will survive, whilethe other cells die).

Expression of proteins in prokaryotes is often carried out in E. coliwith vectors containing constitutive or inducible promoters directingthe expression of either fusion or non-fusion proteins. Fusion vectorsadd a number of amino acids to a protein encoded therein, e.g. to theamino terminus of the recombinant protein. Such fusion vectors typicallyserve three purposes: 1) to increase expression of recombinant protein;2) to increase the solubility of the recombinant protein; and 3) to aidin the purification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein.

As indicated, the expression vectors will preferably contain selectablemarkers. Such markers include dihydrofolate reductase or neomycinresistance for eukaryotic cell culture and tetracyline or ampicillinresistance for culturing in E. coli and other bacteria. Representativeexamples of appropriate host include bacterial cells, such as E. coli,Streptomyces Salmonella typhimurium and certain Bacillus species; fungalcells such as Aspergillus species, for example A. niger, A. oryzae andA. nidulans, such as yeast such as Kluyveromyces, for example K. lactisand/or Puchia, for example P. pastoris; insect cells such as DrosophilaS2 and Spodoptera Sf9; animal cells such as CHO, COS and Bowes melanoma;and plant cells. Appropriate culture mediums and conditions for theabove-described host cells are known in the art.

Vectors preferred for use in bacteria are for example disclosed inWO-A1-2004/074468, which are hereby enclosed by reference. Othersuitable vectors will be readily apparent to the skilled artisan.

Known bacterial promotors suitable for use in the present inventioninclude the promoters disclosed in WO-A1-2004/074468, which are herebyincorporated by reference.

Transcription of the DNA encoding a variant of the present invention byhigher eukaryotes may be increased by inserting an enhancer sequenceinto the vector. Enhancers are cis-acting elements of DNA, usually aboutfrom 10 to 300 bp that act to increase transcriptional activity of apromoter in a given host cell-type. Examples of enhancers include theSV40 enhancer, which is located on the late side of the replicationorigin at by 100 to 270, the cytomegalovirus early promoter enhancer,the polyoma enhancer on the late side of the replication origin, andadenovirus enhancers.

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretation signal may beincorporated into the expressed polypeptide. The signals may beendogenous to the polypeptide or they may be heterologous signals.

A variant of the invention may be expressed in form such that it mayinclude additional heterologous functional regions, for examplesecretion signals. A variant of the invention may also comprise, forexample, a region of additional amino acids, particularly charged aminoacids, added to the N-terminus of the polypeptide for instance toimprove stability and persistence in the host cell, during purificationor during subsequent handling and storage. Also, peptide moieties may beadded to a variant of the invention to facilitate purification, forexample by the addition of histidine residues or a T7 tag.

The variants of the invention, such as proteins of the present inventionor functional equivalents thereof, e.g., biologically active portionsand fragments thereof, can be operatively linked to a non-variantpolypeptide (e.g., heterologous amino acid sequences) to form fusionproteins. A “non-variant polypeptide” in this context refers to apolypeptide having an amino acid sequence corresponding to a proteinwhich is not substantially homologous to a variant asparaginase of theinvention.

Within a fusion protein, the variant of the invention can correspond toa full length sequence or a biologically active fragment of apolypeptide of the invention. In a preferred embodiment, a fusionprotein of the invention comprises at least two biologically activeportions. Within the fusion protein, the term “operatively linked” isintended to indicate that the variant polypeptide and the non-variantpolypeptide are fused in-frame to each other. The non-variantpolypeptide can be fused to the N-terminus or C-terminus of the variantpolypeptide.

For example, in one embodiment, the fusion protein is a fusion proteinin which the variant sequence/s is/are fused to the C-terminus of theGST sequences. Such fusion proteins can facilitate the purification of arecombinant variant according to the invention. In another embodiment,the fusion protein is a variant of the invention containing aheterologous signal sequence at its N-terminus. In certain host cells(e.g., mammalian and yeast host cells), expression and/or secretion of avariant of the invention can be increased through use of a hetereologoussignal sequence.

In another example, the gp67 secretory sequence of the baculovirusenvelope protein can be used as a heterologous signal sequence (CurrentProtocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons,1992). Other examples of eukaryotic heterologous signal sequencesinclude the secretory sequences of melittin and human placental alkalinephosphatase (Stratagene; La Jolla, Calif.). In yet another example,useful prokarytic heterologous signal sequences include the phoAsecretory signal (Sambrook et al., supra) and the protein A secretorysignal (Pharmacia Biotech; Piscataway, N.J.).

A signal sequence can be used to facilitate secretion and isolation of avariant of the invention. Signal sequences are typically characterizedby a core of hydrophobic amino acids, which are generally cleaved fromthe mature protein during secretion in one or more cleavage events. Suchsignal peptides contain processing sites that allow cleavage of thesignal sequence from the mature proteins as they pass through thesecretory pathway. The signal sequence may direct secretion of thevariant, such as from a eukaryotic host into which the expression vectoris transformed, and the signal sequence may then be subsequently orconcurrently cleaved. The variant of the invention may then be readilypurified from the extracellular medium by known methods. Alternatively,the signal sequence can be linked to the variant of interest using asequence, which facilitates purification, such as with a GST domain.Thus, for instance, the sequence encoding the variant of the inventionmay be fused to a marker sequence, such as a sequence encoding apeptide, which facilitates purification of the fused variant of theinvention. In certain preferred embodiments of this aspect of theinvention, the marker sequence is a hexa-histidine peptide, such as thetag provided in a pQE vector (Qiagen, Inc.), among others, many of whichare commercially available. As described in Gentz et al, Proc. Natl.Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine providesfor convenient purification of the fusion protein. The HA tag is anotherpeptide useful for purification which corresponds to an epitope derivedof influenza hemaglutinin protein, which has been described by Wilson etal., Cell 37:767 (1984), for instance.

A fusion protein of the invention may be produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent polypeptide sequences are ligated together in frame inaccordance with conventional techniques, for example by employingblunt-ended or stagger-ended termini for ligation, restriction enzymedigestion to provide for appropriate termini, filling-in of cohesiveends as appropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. In another embodiment, the fusion genecan be synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers, which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,for example, Current Protocols in Molecular Biology, eds. Ausubel et al.John Wiley & Sons: 1992). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide). A variant-encoding nucleic acid can be cloned into such anexpression vector such that the fusion moiety is linked in-frame to thesaid variant.

The terms “functional equivalents” and “functional variants” are usedinterchangeably herein. Functional equivalents according to theinvention are isolated DNA fragments that encode a polypeptide thatexhibits a particular function of a variant as defined herein.Functional equivalents therefore also encompass biologically activefragments and are themselves encompassed within the term “a variant” ofthe invention.

Preferably, a functional equivalent of the invention comprises one ormore of the substitutions described herein. However, a functionalequivalent may comprise one or more modifications in addition to thesubstitutions descrived above.

Functional nucleic acid equivalents may typically contain silentmutations or mutations that do not alter the biological function ofencoded polypeptide. Accordingly, the invention provides nucleic acidmolecules encoding a variant asparaginase protein that contains changesin amino acid residues that are not essential for a particularbiological activity. Such variant proteins differ in amino acid sequencefrom the parent asparaginase sequence from which they are derived yetretain at least one biological activity thereof, preferably they retainat least asparaginase activity. In one embodiment the isolated nucleicacid molecule comprises a nucleotide sequence encoding a protein,wherein the protein comprises a substantially homologous amino acidsequence of at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or more homologous to the parent amino acid sequence (forexample that shown in SEQ ID NO: 3).

As defined herein, the term “substantially homologous” refers to a firstamino acid or nucleotide sequence which contains a sufficient or minimumnumber of identical or equivalent (e.g., with similar side chain) aminoacids or nucleotides to a second amino acid or nucleotide sequence suchthat the first and the second amino acid or nucleotide sequences have acommon domain. For example, amino acid or nucleotide sequences whichcontain a common domain having about 60%, preferably 65%, morepreferably 70%, even more preferably 75%, 80%, 85%, 90%, 95%, 96%, 97%,98% or 99% identity or more are defined herein as sufficientlyidentical.

The skilled person will recognise that changes can be introduced bymutation into the nucleotide sequences according to the inventionthereby leading to changes in the amino acid sequence of the resultingprotein without substantially altering the function of such a protein.

Accordingly, the an asparaginase variant of the invention is preferablya protein which comprises an amino acid sequence at least about 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologousto the parent amino acid sequence, for example that shown in SEQ ID NO:3, and typically also retains at least one functional activity of theparent polypeptide.

Variants of the invention, for example functional equivalents of aprotein according to the invention, can also be identified e.g. byscreening combinatorial libraries of mutants, e.g. truncation mutants,of the protein of the invention for asparaginase activity. In oneembodiment, a variegated library of variants is generated bycombinatorial mutagenesis at the nucleic acid level. A variegatedlibrary of variants can be produced by, for example, enzymaticallyligating a mixture of synthetic oligonucleotides into gene sequencessuch that a degenerate set of potential protein sequences is expressibleas individual polypeptides, or alternatively, as a set of larger fusionproteins (e.g. for phage display). There are a variety of methods thatcan be used to produce libraries of potential variants of thepolypeptides of the invention from a degenerate oligonucleotidesequence. Methods for synthesizing degenerate oligonucleotides are knownin the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et al.(1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477).

In addition, libraries of fragments of the sequence encoding apolypeptide of the invention can be used to generate a variegatedpopulation of polypeptides for screening a subsequent selection ofvariants. For example, a library of coding sequence fragments can begenerated by treating a double stranded PCR fragment of the codingsequence of interest with a nuclease under conditions wherein nickingoccurs only about once per molecule, denaturing the double stranded DNA,renaturing the DNA to form double stranded DNA which can includesense/antisense pairs from different nicked products, removing singlestranded portions from reformed duplexes by treatment with S1 nuclease,and ligating the resulting fragment library into an expression vector.By this method, an expression library can be derived which encodesN-terminal and internal fragments of various sizes of the protein ofinterest.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations of truncation, and forscreening cDNA libraries for gene products having a selected property.The most widely used techniques, which are amenable to high through-putanalysis, for screening large gene libraries typically include cloningthe gene library into replicable expression vectors, transformingappropriate cells with the resulting library of vectors, and expressingthe combinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a techniquewhich enhances the frequency of functional mutants in the libraries, canbe used in combination with the screening assays to identify variants ofa protein of the invention (Arkin and Yourvan (1992) Proc. Natl. Acad.Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).

Fragments of a polynucleotide according to the invention may alsocomprise polynucleotides not encoding functional polypeptides. Suchpolynucleotides may function as probes or primers for a PCR reaction.

Nucleic acids according to the invention irrespective of whether theyencode functional or non-functional polypeptides can be used ashybridization probes or polymerase chain reaction (PCR) primers. Uses ofthe nucleic acid molecules of the present invention that do not encode apolypeptide having asparaginase activity include, inter alia, (1) insitu hybridization (e.g. FISH) to metaphase chromosomal spreads toprovide precise chromosomal location of an asparaginase-encoding gene asdescribed in Verma et al., Human Chromosomes: a Manual of BasicTechniques, Pergamon Press, New York (1988); (2) Northern blot analysisfor detecting expression of asparaginase mRNA in specific tissues and/orcells; and (3) probes and primers that can be used as a diagnostic toolto analyse the presence of a nucleic acid hybridizable to such a probeor primer in a given biological (e.g. tissue) sample.

Variants of a given parent asparaginase enzyme can be obtained by thefollowing standard procedure:

-   -   Mutagenesis (error-prone, doped oligo, spiked oligo)    -   Primary Screening    -   Identification of an improved variant (for example in relation        to specific activity)    -   Maintenance (for example in glycerol culture, LB-Amp plates,        Mini-Prep)    -   Streaking out on another assay plate-secondary screening    -   DNA Sequencing    -   Transformation in, for example Aspergillus    -   Cultivation, for example in 100 ml scale, purification, DSC

In one embodiment the invention relates to a method of producing anasparaginase polypeptide variant according to the invention, whichmethod comprises:

-   a) selecting a parent asparaginase polypeptide;-   b) substituting at least one amino acid residue corresponding to any    of    -   41, 53, 62, 63, 64, 66, 70, 71, 73, 74, 76, 77, 88, 90, 91, 101,        102, 103, 104, 106, 107, 108, 109, 111, 119, 122, 123, 132, 140,        142, 143, 145, 161, 162, 163, 164, 168, 169, 170, 195, 211, 213,        214, 215, 216, 217, 218, 219, 220, 228, 232, 233, 234, 235, 262,        267, 268, 269, 270, 271, 272, 273, 293, 295, 297, 298, 299, 300,        301, 302, 303, 304, 310, 314, 317, 318, 319, 321, 323, 324, 325,        327, 328, 329, 330, 331, 332, 333, 334, 335 or 371

said positions being defined with reference to SEQ ID NO: 3;

-   c) optionally substituting one or more further amino acids as    defined in b);-   d) preparing the variant resulting from steps a)-c);-   e) determining the specific activity at at least one pH and/or the    pH optimum of the variant; and-   f) selecting a variant having an increased specific activity at at    least one pH in comparison to the parent asparaginase polypeptide    and/or increased pH optimum in comparison to the parent asparaginase    polypeptide, thereby to produce an asparaginase polypeptide variant.

In a preferred embodiment in the method of producing an asparaginasepolypeptide variant according to the invention, the parent asparaginasepolypeptide has the sequence set out in SEQ ID NO: 3.

More preferably in step b) of the method according to the invention atleast one amino acid residue corresponding to any of

41, 53, 63, 64, 66, 73, 74, 76, 77, 88, 90, 91, 101, 106, 111, 119, 122,123, 132, 140, 145, 161, 170, 195, 211, 218, 228, 232, 233, 262, 267,293, 295, 297, 299, 300, 301, 303, 304, 310, 314, 317, 321, 324, 330,332 or 333, 371,is substituted, said positions being defined with reference to SEQ IDNO: 3 and wherein the parent polypeptide has at least 80% homology withSEQ ID NO: 3.

Even more preferably in step b) of the method according to the inventionthe substituted amino acid residue corresponds to one or more of Ile atposition 41, Tyr at position 53, Gly or Val or Ser at position 63, Proat position 64, Pro at position 66, Lys at position 73, Ala at position74, Thr at position 76, Ile at position 77, Tyr or Pro at position 88,Val at position 90, Glu at position 91, Val at position 101, Pro atposition 106, Gly at position 111, Asn at position 119, His at position122, Ala at position 123, Ser at position 132, Asn at position 140, Serat position 145, Leu at position 161, Thr at position 170, Asp atposition 195, Ser at position 211, Val at position 218, His at position228, Val at position 232, Val at position 233, Cys or His at position262, Tyr at position 267, Ser or Val at position 293, Ser at position295, Ser at position 297, Ser at position 299, Ile at position 300, Proat position 301, Ser at position 303, Thr at position 304, Val atposition 310, Asp at position 314, Ile at position 317, Thr at position321, Gly at position 324, Ser at position 330, Ala at position 332, Gluat position 333 or Met at position 371 said positions being defined withreference to SEQ ID NO: 3.

In one embodiment of the process of the invention in step e) thespecific activity is determined at a pH between 4 and 8. In anotherembodiment of step e), prior to determining the specific activity at atleast one pH and/or the pH optimum of the variant, the ratio, at aspecific temperature, between the asparaginase activity at pH 7 and theasparaginase activity at pH 5 of the variant may be measured and avariant may be selected wherein said ratio is higher than that of theparent asparaginase polypeptide.

In another embodiment of the process of the invention in step f) avariant is selected having an increased specific activity at at leastone pH, preferably at a pH between 4 and 8, in comparison to the parentpolypeptide and/or having an increased pH optimum in comparison to theparent polypeptide. Preferably the variant has an increased specificactivity at at least one pH, preferably at a pH between 4 and 8, incomparison to the parent polypeptide and an increased pH optimum incomparison to the parent polypeptide. In another embodiment of theprocess of the invention in step f) a variant is selected having anincreased specific activity at at least one pH, preferably at a pHbetween 4 and 8, in comparison to the parent polypeptide.

In another embodiment, the invention features cells, e.g., transformedhost cells or recombinant host cells that contain a nucleic acidencompassed by the invention. A “transformed cell” or “recombinant cell”is a cell into which (or into an ancestor of which) has been introduced,by means of recombinant DNA techniques, a nucleic acid according to theinvention. Both prokaryotic and eukaryotic cells are included, e.g.,bacteria, fungi, yeast, and the like, especially preferred are cellsfrom filamentous fungi, in particular Aspergillus niger.

A host cell can be chosen that modulates the expression of the insertedsequences, or modifies and processes the product encoded by theincorporated nucleic acid sequence in a specific, desired fashion. Suchmodifications (e.g., glycosylation) and processing (e.g., cleavage) ofprotein products may facilitate optimal functioning of the encodedprotein.

Various host cells have characteristic and specific mechanisms forpost-translational processing and modification of proteins and geneproducts. Appropriate cell lines or host systems familiar to those ofskill in the art of molecular biology and/or microbiology can be chosento ensure the desired and correct modification and processing of theforeign protein expressed. To this end, eukaryotic host cells thatpossess the cellular machinery for proper processing of the primarytranscript, glycosylation, and phosphorylation of the gene product canbe used. Such host cells are well known in the art.

Host cells also include, but are not limited to, mammalian cell linessuch as CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and choroidplexus cell lines.

If desired, a stably transfected cell line can produce a variantaccording to the invention. A number of vectors suitable for stabletransfection of mammalian cells are available to the public, methods forconstructing such cell lines are also publicly known, e.g., in Ausubelet al. (supra).

The present invention further discloses a composition comprising theasparaginase variants according to the invention. The composition mayoptionally comprise other ingredients such as e.g. other enzymes. Theasparaginase variants according to the invention or compositionscomprising said asparaginases can be used in the production of a foodproduct. In one embodiment of the invention the asparaginase variants orcompositions according to the invention can be used to reduce the amountof acrylamide formed in a thermally processed food product based on anasparagine-containing raw material. They can, for example, be used in aprocess for the production of a food product involving at least oneheating step, comprising adding one or more asparaginase enzymes to anintermediate form of said food product in said production processwhereby the enzyme is added prior to said heating step in an amount thatis effective in reducing the level of asparaginase that is present insaid intermediate form of said food product. Such process is disclosedin WO04/030468 which process and all its preferences are hereinincorporated by reference. Also in WO04/026043 suitable processes aredescribed wherein the asparaginase according to the invention could beused. The processes disclosed in WO04/026043 and all preferencesdisclosed are herein incorporated by reference.

An intermediate form of the food product is defined herein as any formthat occurs during the production process prior to obtaining the finalform of the food product. The intermediate form may comprise theindividual raw materials used and/or mixture thereof and/or mixtureswith additives and/or processing aids, or subsequently processed formthereof. For example, for the food product bread, the intermediate formscomprise for example wheat, wheat flour, the initial mixture thereofwith other bread ingredients such as for example water, salt, yeast andbread improving compositions, the mixed dough, the kneaded dough, theleavened dough and the partially baked dough. For example for severalpotato-based products, dehydrated potato flakes or granules areintermediate products, and corn masa is an intermediate product fortortilla chips.

The food product may be made from at least one raw material that is ofplant origin, for example potato, tobacco, coffee, cocoa, rice, cereal,for example wheat, rye corn, maize, barley, groats, buckwheat and oat.Wheat is here and hereafter intended to encompass all known species ofthe Triticum genus, for example aestivum, durum and/or spelta. Also foodproducts made from more than one raw material or intermediate areincluded in the scope of this invention, for example food productscomprising both wheat (flour and/or starch) and potato. Examples of foodproducts in which the process according the invention can be suitablefor are any flour based products—for example bread, pastry, cake,pretzels, bagels, Dutch honey cake, cookies, gingerbread, gingercake andcrispbread-, and any potato-based products—for example French fries,pommes frites, potato chips, croquettes.

Raw materials as cited above are known to contain substantial amounts ofasparagine which is involved in the formation of acrylamide during theheating step of the production process. Alternatively, the asparaginemay originate from other sources than the raw materials e.g. fromprotein hydrolysates, such as yeast extracts, soy hydrolysate, caseinhydrolysate and the like, which are used as an additive in the foodproduction process. A preferred production process is the baking ofbread and other baked products from wheat flour and/or flours from othercereal origin. Another preferred production process is the deep-fryingof potato chips from potato slices.

Preferred heating steps are those at which at least a part of theintermediate food product, e.g. the surface of the food product, isexposed to temperatures at which the formation of acrylamide ispromoted, e.g. 110° C. or higher, 120° C. or higher temperatures. Theheating step in the process according to the invention may be carriedout in ovens, for instance at a temperature between 180-220° C., such asfor the baking of bread and other bakery products, or in oil such as thefrying of potato chips, for example at 160-190° C.

In another aspect, the invention provides food products obtainable bythe process of the invention as described hereinbefore or by the use ofthe novel asparaginase as described hereinbefore to produce foodproducts. These food products are characterized by significantly reducedacrylamide levels in comparison with the food products obtainable byproduction processes that do not comprise adding one or more enzymes inan amount that is effective in reducing the level of amino acids whichare involved in the formation of acrylamide during said heating step.The process according to the invention can be used to obtain a decreaseof the acrylamide content of the produced food product preferably morethan 50%, more preferably more than 20%, even more preferably 10% andmost preferably more than 5% compared to a food product obtained withthe conventional process.

An additional application for the asparaginase variants according to theinvention is to be employed in the therapy of tumours. The metabolism oftumour cells requires L-asparagine, which can quickly be degraded byasparaginases. The asparaginase according to the invention can also beused as an adjunct in treatment of some human leukaemia. Administrationof asparaginase in experimental animals and humans leads to regressionof certain lymphomas and leukemia. Therefore in one embodiment theinvention relates to asparaginases or a composition according to theinvention for use as medicament, e.g. in the treatment of tumors, e.g.in the treatment of lymphomas or leukaemia in animals or humans.

Asparaginase variants according to the invention may conveniently beproduced in microorganisms. In the above processes, it is advantageousto use asparaginases that are obtained by recombinant DNA techniques.Such recombinant enzymes have a number of advantages, such as productionat a low cost price, high yield, free from contaminating agents such asbacteria or viruses, but also free from bacterial toxins orcontaminating other enzyme activities.

The invention is hereinafter illustrated by the following non-limitingexamples.

EXAMPLES Materials & Methods

Asparaginase Assay in Order to Measure pH Dependence in Range pH=4 topH=9

The asparaginase activity was measured using L-asparagine as substrate.The amount of ammonia that was liberated by the action of the enzyme wasmeasured according to the Berthelot reaction. Ready-to-use reagentsphenolnitroprusside and alkaline hypoclorite were obtained from Sigma.100 μl enzyme sample was mixed with 2000 μl 100 mM L-asparagine in amixture of 50 mM citric acid and 50 mM sodium pyrophosphate buffer ofthe desired pH. After incubation at 37° C. for 30 minutes the reactionwas stopped by adding 400 μl 25% trichloroacetic acid, whereafter 2500μl water was added. During the incubation the temperature was fixed at37° C. unless indicated otherwise.

It should be understood by a person skilled in the art that enzymedosing was chosen in such a way that after incubation under the aboveconditions a signal was obtained significantly above the background butstill within a range where the signals obtained are proportional to theamount of enzyme added. Preferably the reaction was zero order.

After stopping the reaction, 4 μl of the incubation mixture was added to156 μl water. Subsequently 34 μl phenol/nitroprusside solution (SigmaP6994) and 34 μl alkaline hypochlorite solution (Sigma A1727) wereadded. After 676 seconds of incubation at 37° C., the extinction wasmeasured at 600 nm. Readings were corrected for the background signal byincluding the appropriate blanks. A sample with (TCA) inactivated enzymewas used as a blank. The assays were run on an autoanalyzer e.g. aKonelab Arena 30 (Thermo Scientific). The activity was determined usinga calibration line made up by plotting the measured absorbance at 600 nmversus the known ammonium sulphate concentrations of a standard series.Activity was expressed in units, where one unit is defined as the amountof enzyme required to liberate one micromole of ammonia fromL-asparagine per minute under defined assay conditions.

Asparaginase Assay in Order to Measure pH Dependence in Range pH=4 topH=8

The method was executed in the same way as to the method described abovefor measurement of pH dependence of the activity for the range pH=4 topH=9, with the difference that 100 μl enzyme sample was mixed with 2000μl 100 mM L-asparagine in a 50 mM phosphate/citric acid buffer of thedesired pH.

Asparaginase Manual Assay in Order to Measure Activity at pH=5 and pH=7

The assay was performed e.g. in microtiterplates (MTP's) or tubes. Toidentify asparaginases with a shifted pH-activity profile, activity wasmeasured at pH=5 and pH=7. 10 μl enzyme sample was mixed with 190 μl 100mM L-asparagine in 100 mM citric acid buffer pH 5.0 or 100 mM phosphatebuffer pH 7.0. After incubation at room temperature and for 1 hr thereaction was stopped by adding 100 μl 12.5% trichloroacetic acid. Theenzyme dosing was chosen in such a way that after 1 hour incubation atroom temperature, a signal was obtained significantly above thebackground. After stopping the reaction, 95 μl water was added to 8 μlof the incubation mixture. Subsequently, 70 μl phenol/nitroprussidesolution (Sigma P6994) and 70 μl alkaline hypochlorite solution (SigmaA1727) were added. After 60 minutes of incubation at room temperature,the extinction was measured at 620 nm. Readings were corrected for thebackground signal by including the appropriate blanks e.g. inactivatedsample and/or supernatant from fermentation samples of empty hoststrains. Empty strain indicates a host strain which has not beentransformed to contain the asparaginase gene. The activity wasdetermined using a calibration line made up by plotting the measuredabsorbance at 620 nm versus the known ammonium sulphate concentrationsof a standard series. Activity is expressed in units, where one unit isdefined as the amount of enzyme required to liberate one micromole ofammonia from L-asparagine per minute under defined assay conditions.

In all assays the activity of the asparaginase samples were expressed inunit/ml.

Example 1 Fermentation, Isolation and Purification of AsparaginasesAccording to the Invention

Asparaginases of the invention were obtained by the construction ofexpression plasmids containing a DNA sequence encoding the asparaginaseof the invention, transforming an Aspergillus niger strain with theplasmid and growing the Aspergillus niger strains as described inWO2004/030468.

After growing Aspergillus niger containing the proper expressionplasmids cell free supernatants were prepared by centrifugation of thefermentation broth at 5000×g for 30 minutes at 4° C. If necessary thesupernatants were filtered further over a Miracloth filter (Calbiochemcat#475855) and a GF/A Whatmann Glass microfiber filter (150 mm {acuteover (Ø)}), respectively, to remove any solids. To remove any fungalmaterial the supernatants could be adjusted to pH=5 with 4N KOH andsterile filtrated over a 2 μm (bottle-top) filter with suction. Thesupernatants were stored until use at 4° C. or frozen at −20° C. ifnecessary.

In case impurities were more than 60% w/w asparaginase were purified byanion ion-exchange chromatography starting from cell free supernatantsor ccUF desalted via a PD-10 column (Amersham Biosciences). The desaltedmaterial was applied to a Mono-Q or Q-Sepharose column equilibrated in20 mM histidine buffer pH 5.96. After extensive washing theasparaginases were eluted from the column using a gradient from 0 to 1MNaCl.

The purity of the supernatant fractions containing the asparaginaseactivity or of the purified asparaginase fractions (determined in mgprotein/ml) was checked by analytical size-exclusion chromatography(HP-SEC: High Performance Size Exclusion Chromatography, TSKgel3000SW-XL, column 300*7,8 mm; MW range 10-300 kDa, 100 mM phosphatebuffer pH7 and pH5.96). All flows were 1 ml/min (except for sampleinjection on the Q-Sepharose column, which was at 5 ml/min). Detectionof eluted proteins was done at 280 nm. The concentration of the elutedAspergillus niger wild type asparaginase was calculated from theextinction at 280 nm (A280) using a molar extinction coefficient of10240 M⁻¹·cm⁻¹ (A280^(1cm,1mg/ml)=0.28, wherein A280^(1cm,1mg/ml) is theextinction at 280 nm measured with a path length of 1 cm and at aconcentration of pure protein of 1 mg/ml). Measurement of the A280 wasperformed in a Uvikon XL Secomam spectrophotometer (Beun de Ronde,Abcoude, The Netherlands). For asparaginases corresponding to ASN15 abdASN 16 the same extinction coefficient as that of the Aspergillus nigerwild type asparaginase was used. In case of impurities absorbing at 280nm the asparaginase concentration was corrected based on the HP-SECchromatogram by multiplying the measured A280 of the asparaginase sampleby the ratio of the area under the asparaginase peak and the total areaof the peaks absorbing at 280 nm. When the asparaginase peaks was notclearly separated from other peaks the peak heights instead of peakareas were taken.

For asparaginases corresponding to ASN 01 to ASN14 the asparaginasecontent (determined in mg protein/ml) can be determined by PAA-SDS gelelectrophoresis using NuPAGE® Novex 4-12% Bis-Tris 12 well gels(Invitrogen, NP0322BOX). 1 μl of culture supernatant was incubated with1 μl 10×NuPAGE® Sample Reducing Agent (Invitrogen, NP0004), 2.5 μl4×NuPAGE LDS Sample Buffer (Invitrogen, NP0007) and 5.5 μl milliQ waterfor 10 minutes at 70° C. The resulting reduced sample was loaded on thegel. The SeeBlue® Plus2 prestained standard (Invitrogen, LC5925) wasused as size marker. In addition, 0.5 μg of BSA (Sigma A9418) was loadedas calibrator for the amount of protein. The gels were run in NuPAGE®MES SDS running buffer (Invitrogen, NP0002), containing NuPAGE®Antioxidant (Invitrogen, NP0005) for 35 minutes at 200 V. Followingelectrophoresis, the gels were fixed for 2×30 minutes in Fix solution(7% HAc (v/v) and 10% ethanol (v/v)), stained over night with SYPRO Rubyprotein gel stain (Invitrogen S12000) and de-stained in Fix solution for2×30 minutes. Subsequently, the gels were washed with demineralisedwater and scanned with the Typhoon 9200 scanner (GE Healthcare). Thepeak volume was calculated using Image Quant TLv2003.02 software and theprotein concentrations were calculated based on the BSA protein band.

Example 2 Performance of the Variant Asparaginases According to theInvention

A random libraray of A. niger asparaginase mutants (wherein the parentpolypeptide was that according to SEQ ID NO: 3) was screened for mutantswith a changed pH-activity profile. In order to find mutants withimproved activity at more alkaline pH, the activity of the asparaginasemutants was determined at pH=5 and pH=7. Subsequently the ratio betweenthe activity at pH=7 and the activity at pH=5 was determined. This ratiois shown in table 1. A higher ratio indicates a shift of the pH-activityprofile towards pH=7.

TABLE 1 Third column: Ratio between activity at pH = 7 and activity atpH = 5 for selected mutants. Wild type (WT) is A. niger asparaginase(WO2004/030468). Fourth column: The pH shift of the alkaline limb of thepH-activity profile represented by the pH at which the mutant stillexhibits 50% of its maximal catalytic activity. The pH-activity profileswere determined at 37° C. using cell-free supernatants. Activity wasmeasured in the range pH = 4 to pH = 8, using a phosphate/citric acidbuffer system. Amino acid Ratio between Alkaline substitution activityat pH at if compared pH = 7 and which still Amino with SEQ ID activityat 50% activity acid Variant NO: 3 pH = 5 is observed sequence WT 0.426.7 SEQ ID NO: 3 ASN01 D63G + G132S 1.21 8.0 SEQ ID NO: 4 ASN02 D63G +D111G + 1.25 8.1 SEQ ID R122H NO: 5 ASN03 D63V + T300I 0.60 7.3 SEQ IDNO: 6 ASN04 S64P + I310V 0.57 7.2 SEQ ID NO: 7 ASN05 T41I + S66P + 0.907.8 SEQ ID V371M NO: 8 ASN06 A76T + A101V 0.88 7.6 SEQ ID NO: 9 ASN07V77I + V123A + 0.54 7.1 SEQ ID E314D NO: 10 ASN08 S88Y 0.63 7.4 SEQ IDNO: 11 ASN09 S88P + I161L + 0.73 7.6 SEQ ID R262C NO: 12 ASN10 D140N0.73 7.4 SEQ ID NO: 13 ASN11 D91E + A170T + 0.83 7.5 SEQ ID R262H NO: 14ASN12 L90V + K119N + 0.62 7.3 SEQ ID Y228H + R262C NO: 15 ASN13 F53Y +K119N 0.48 6.9 SEQ ID NO: 16 ASN14 G195D + A293V 0.75 7.4 SEQ ID NO: 17

Mutants with a higher ratio than the wild type A. niger asparaginasewere further tested to establish to which extent the pH activity profilewas shifted to alkaline pH. For these mutants a full pH-activity profilewas measured and it was shown that in particular the alkaline limb ofthe pH-activity profile has shifted to higher pH. The pH at which thealkaline limb of the pH-activity profile shows 50% of the maximalactivity of a mutant at its pH optimum is taken as an indicator for ashift of the alkaline limb of the pH activity profile compared to wildtype (table 1). A shift to a higher pH indicates a higher activity undermore alkaline conditions. Such mutants are in particular beneficial inapplications that require more alkaline condition.

When selecting for mutants with a lower ratio between activity at pH=7and activity at pH=5 it is observed that the alkaline limb of thepH-activity profile shifts to a lower pH (table 2).

TABLE 2 Ratio between activity at pH = 7 and activity at pH = 5 forselected mutants. Wild type (WT) is A. niger asparaginase(WO2004/030468). The pH-activity profiles were determined at 37° C.using cell-free supernatants. Activity was measured in the range pH = 4to pH = 8, using a phosphate/citric acid buffer system. Amino acid Ratiobetween Alkaline substitution activity at pH at if compared pH = 7 andwhich still Amino with SEQ ID activity at 50% activity acid Variant NO:3 pH = 5 is observed sequence WT 0.42 6.7 SEQ ID NO: 3 ASN15 T73K +S74A + 0.37 6.5 SEQ ID A293S NO: 18 ASN16 T73K + S74A + 0.08 5.9 SEQ IDE106P + A293S + NO: 19 G297S + T299S + Q319A + M321T + V324GThe pH-Activity Dependence and the pH Optimum

The pH dependence of the asparaginase activity for the mutants wasdetermined in 50 mM phosphate/citrate buffer for the pH range pH=4 topH=8 using cell-free supernatants. The pH at which the highest activitywas observed for a mutant is called the pH optimum for the said mutant.In tables 3 and 4 the maximum activity observed for a mutant is set to100% and activities of said mutant at other pH values are shown aspercentage of the maximum activity observed for said mutant. In table 3the pH-activity profile was determined for the pH range pH=4 to pH=8using the phosphate/citric acid buffer system. In table 4 thepH-activity profile was determined for the pH range pH=4 to pH=9 usingthe pyrophosphate/citric acid buffer system.

TABLE 3 The pH dependence of the asparaginase activity for the mutantscompared to wild type (wt) A. niger asparaginase (WO2004/030468). Thehighest activity that was observed for each asparaginase was set to100%. Activity was determined using cell-free supernatant at 37° C. inthe phosphate/citric acid buffer system. Amino acid substitution ifcompared with pH = pH = pH = pH = pH = Variant SEQ ID NO: 3 4 5 6 7 8ASN03 D63V + T300I 79% 100% 91% 60% 16% ASN04 S64P + I310V 99% 100% 84%57% 16% ASN05 T41I + S66P + 88%  99% 100%  89% 40% V371M ASN07 V77I +V123A + 100%  100% 81% 54% 15% E314D ASN09 S88P + I161L + 89% 100% 93%73% 33% R262C ASN10 D140N 77%  95% 100%  69% 11% ASN12 L90V + K119N +91% 100% 87% 62% 19% Y228H + R262C ASN13 F53Y + K119N 98% 100% 76% 48%18% WT WT 100%   99% 72% 43% 14%

TABLE 4 The pH dependence of the asparaginase activity for the mutantscompared to wild type (wt) A. niger asparaginase (WO2004/030468). Thehighest activity that was observed for each asparaginase was set to100%. Activity was determined with cell-free supernatants at 37° C.using the pyrophosphate/citric acid buffer system. Amino acidsubstitution if compared Vari- with SEQ pH = pH = pH = pH = pH = pH =ant ID NO: 3 4 5 6 7 8 9 ASN01 D63G + 72%  83% 97% 100%  50% 0% G132SASN02 D63G + 71%  80% 93% 100%  56% 1% D111G + R122H ASN06 A76T + 91%100% 98% 88% 22% 1% A101V ASN08 S88Y 96% 100% 86% 66% 23% 1% ASN11D91E + 74%  83% 100%  94% 11% 0% A170T + R262H ASN14 G195D + 77% 100%86% 75%  7% 0% A293V WT WT 100%  100% 72% 43%  9% 0%

Apart from a shift of the alkaline limb of the pH-activity profile thereis also a shift of the pH optimum towards higher pH. Both the mutantscontaining the mutation D63G show a shift of the pH optimum to pH=7. TheD63G mutants contain additional muations. However these additionalmuations are different in each D63G mutant, while the pH-activityprofiles are almost identical. Therefore D63G seems to cause theobserved shift of the pH-activity profile. The pH optimum of mutantD140N, the mutant containing A170T, and the mutant containing themutation S66P is shifted to pH=6.

The remaining mutants exhibit a more explicit pH optimum at pH=5compared to wild type. Tables 3 and 4 show clearly the shift of thealkaline limb of the pH-activity profile towards higher pH resulting ina broader pH-activity profile with on particular increased relativeactivity for the range pH=6 to pH=8, while at the same time asubstantial activity is also maintained in the acidic region pH=4 topH=6.

Specific Activity as a Function of pH

The specific activity of the asparaginase variants was determined atpH=4, pH=5, pH=6, pH=7, pH=8 at 37° C. in 50 mM phosphate/citrate bufferusing cell-free supernatants.

TABLE 5 The specific activity (measured by dividing the activity of asample (in units/ml) by mg/ml asparaginase present in the sample) of thevariants relative to wild type A. niger asparaginase (WO2004/030468) atthe indicated pH values using asparagine as a substrate. For each pH thewild type specific activity was set to 100% and the activity of themutants calculated relative to wild type asparaginase. When activity ofthe mutants was below 100%, the activity was omitted from the table.Activity was determined at 37° C. The amount of asparaginase protein inthe cell-free supernatants was determined by performing PAA-SDS gelelectroforesis experiments and scanning of the gels as is described inmaterial and methods. For T73K + S74A + A293S and T73K + S74A + A293S +E106P + G297S + T299S + Q319A + M321T + V324G the asparaginase proteinconcentration was derived from an A280 measurement applying a correctionfor any impurities based on HP-SEC chromatography. Amino acidsubstitution if compared with Variant SEQ ID NO: 3 pH 4 pH 5 pH 6 pH 7pH 8 WT WT 100%  100% 100% 100% 100% ASN01 D63G + G132S 86%  96% 156%254% 408% ASN02 D63G + D111G + 87% 101% 156% 282% 543% R122H ASN03S64P + I310V 89%  91% 106% 120% 105% ASN05 T41I + S66P + V371M 164% 187% 260% 392% 527% ASN06 A76T + A101V 64%  71%  97% 148% 227% ASN08S88Y 83%  87% 107% 129% 159% ASN09 S88P + I161L + R262C 135%  152% 195%257% 350% ASN11 D91E + A170T + 46%  51%  66%  91% 133% R262H ASN12L90V + K119N + 82%  92% 111% 132% 120% Y228H + R262C ASN13 F53Y + K119N185% 189% 199% 214% 235% ASN14 G195D + A293V 79%  93% 110% 159% 158%ASN15 T73K + S74A + A293S 166%  173% 154% 151% 142% ASN16 T73K + S74A +106%  189% 125%  37%  8% A293S + E106P + G297S + T299S + Q319A + M321T +V324G

Table 5 shows that the specific activity of the mutants at pH=6, pH=7and pH=8 has been substantially improved compared to wild typeasparaginase. In particular mutants T73K+S74A+A293S, T41I+S66P+V371M,S88P+I161L+R262C and F53Y+K119N are very useful because they show ahigher activity over the whole pH range pH=4 to pH=8. MutantT73K+S74A+A293S+E106P+G297S+T299S+Q319A+M321T+V324G is more active inthe acidic pH region pH=4 to pH=6.

Temperature Optimum

In order to verify the dependence of the activity on the temperature theactivity was measured at different temperatures. In one assay the enzymereaction was stopped after 10 minutes, in a second assay the reactionwas stopped after 30 minutes. The enzyme dosing in the 30 minutes assaywas one third of dosing in the 10 minutes assay. If the enzymes arestable under the applied conditions the observed activity should besimilar. In case inactivation occurs one expects activity to decreaseafter longer assay time. Results are shown in table 6.

TABLE 6 Temperature dependence of the activity. Assay was carried at pH= 5 in 50 mM phosphate/citric acid buffer. The enzyme dosing in 30minutes assay was one third of dosing in 10 minutes assay. The highestactivity at 10 minutes incubation time was set to 100%. Amino acidsubstitution if compared with 50° C. 60° C. 70° C. Variant SEQ ID NO: 310 min 30 min 10 min 30 min 10 min 30 min WT WT 100%  98% 98% 97% 62% 58% ASN01 D63G + G132S 67% 70% 89% 95% 100%  107% ASN02 D63G + D111G +70% 72% 87% 91% 99% 106% R122H ASN04 T41I + S66P + 77% 79% 93% 98% 100% 102% V371M ASN09 S88P + I161L + 84% 87% 100%  100%  95%  95% R262C ASN08S88Y 85% 93% 100%  104%  85%  88%

Table 6 indicates that the stability of the variants is very similar towild type Aspergillus niger asparaginase. Mutants show no reduction inactivity after 30 minutes incubation compared to 10 minutes even at 70°C., which indicates mutants are stable at least for 30 minutes at 70° C.Surprisingly it is observed that the temperature optimum of the mutantsis shifted to higher temperature. For wild type the temperature optimumis at 50° C. considering the temperatures which are tested. For mutantsS16A+D63G+G132S, D63G+D111G+R122H and T41I+S66P+V371M it has shifted to70° C. Such properties are in particular useful in applications thatrequire asparaginases working at elevated temperatures.

1. A variant of a parent polypeptide having asparaginase activity,wherein the variant has an amino acid sequence which, when aligned withthe sequence set out in SEQ ID NO: 3, comprises an amino residuecorresponding to any of amino acids Tyr or Leu at position 53; Ala, Asn,Asp or Pro at position 64; Asn, Lys or Pro at position 66; Ala or Ser atposition 70; Ala, Ser, Asn or Glu at position 71; His, Ser, Asn, Asp,Gln, Glu, Arg or Lys at position 73; Ala or Val at position 74; Tyr, Proor Glu at position 88; Val, Tyr or Phe at position 90; Ser, Asn or Gluat position 91; Ser, Arg or Lys at position 102; Leu, Ile, Phe, Met orThr at position 103; Ser, Asn or Glu at position 107; Arg, Asp, Gly, Asnor Ser at position 109; Gly, Ser, Thr or H is at position 111; Val, Leu,Phe or Met at position 161; Gly or Ser at position 164; Ala, Gly or Thrat position 168; Ser, Val, Met, Ile, Asn or Gln at position 211; Ser orHis at position 214; Ser or Thr at position 215; Ser, Thr, Val, Leu orPhe at position 216; Ala, Ser, Asn, Gln or Glu at position 219; Ala orSer at position 220; Ala, Ser, Asn or His at position 228; Gly, Ser,Asp, or Ile at position 235; Cys or His at position 262; Tyr at position267; Asn or Glu at position 271; Asn or Ser at position 295; Gln, Asn,His, Trp Val, Ile or Tyr at position 302; Gly, Leu, Lys, Glu Asp, Ile,Ala, Tyr, Phe or Ser at position 303; Ala, Val, Met or Thr at position310; Gly, Ala, Ser, Asn, Asp, Gln or His at position 314; Ile atposition 317; Ala, Thr, Leu, Arg, Val, Ile, Tyr or His at position 319;Ser, Thr, His, Arg, Lys or Ala at position 321; Ile, Val or Ser atposition 323; Met, Ala, Gly or Pro at position 324; Ala, Ser, Thr, Asp,Glu or Trp at position 325; Met, Ile, Pro, Tyr, Ser, Ala, Arg, Val orThr at position 327; Ser, Thr or Ile at position 328; Ala, Thr, Leu orGly at position 329; Asp, Glu, Arg, Gln, Val, Pro, Ser, Tyr, Thr, Ile orLeu at position 330; Ala, Thr, Gly, Asn, Asp, Glu, Lys or Arg atposition 331; Ala, Asn, Ser, Gly, Glu, Lys, Pro or Gln at position 332;Glu, Asp, Ser, Ile, Phe, Ala, Gly or Lys at position 333; Gly, Thr, Asp,Glu, Pro, Val or Ile at position 334; and Thr, Gly or Asp at position335, said positions being defined with reference to SEQ ID NO:
 3. 2. Thevariant of claim 1, comprising Tyr at position 53; Pro at position 64;Pro at position 66; Lys at position 73; Ala at position 74; Tyr or Proat position 88; Val at position 90; Glu at position 91; Gly at position111; Leu at position 161; Ser at position 211; His at position 228; Cysor His at position 262; Tyr at position 267; Ser at position 295; Ser atposition 303; Val at position 310; Asp at position 314; Ile at position317; Thr at position 321; Gly at position 324; Ser at position 330; Alaat position 332; or Glu at position 333, said positions being definedwith reference to SEQ ID NO:
 3. 3. The variant of claim 1, comprisingPro at position 66, said position being defined with reference to SEQ IDNO:
 3. 4. The variant according to claim 1 which has at least 95%homology with SEQ ID NO:
 3. 5. The variant according to claim 1 whichhas a specific activity, which is higher than that of the parentpolypeptide measured at the same pH.
 6. The variant according to claim1, wherein the variant has a pH optimum which is higher than that of theparent polypeptide.
 7. The variant according to claim 1 which furthercomprises additional substitution of amino acid residues selected froman amino residue corresponding to any of amino acids 41, 62, 63, 76, 77,101, 104, 106, 108, 119, 122, 123, 132, 140, 142, 143, 145, 162, 163,169, 170, 195, 213, 217, 218, 232, 233, 234, 268, 269, 270, 272, 273,293, 297, 298, 300, 301, 304, or 371, said positions being defined withreference to SEQ ID NO:3.
 8. The variant according to claim 7 whichcomprises from 1 to 5 additional substitutions.
 9. An isolated nucleicacid encoding the variant according to claim
 1. 10. A nucleic acidconstruct comprising the nucleic acid sequence of claim 9 operablylinked to one or more control sequences capable of directing theexpression of an asparaginase in a suitable expression host.
 11. Arecombinant expression vector comprising the nucleic acid construct ofclaim
 10. 12. A recombinant host cell comprising the expression vectorof claim
 11. 13. A method for producing an asparaginase comprisingcultivating the host cell of claim 12 under conditions conducive toproduction of the asparaginase and recovering the asparaginase.
 14. Amethod of producing an asparaginase polypeptide variant according toclaim 1, which method comprises: a) selecting a parent asparaginasepolypeptide; b) substituting at least one amino acid residuecorresponding to any positions 53, 64, 66, 70, 71, 73, 74, 88, 90, 91,102, 103, 107, 109, 111, 161, 164, 168, 211, 214, 215, 216, 219, 220,228, 235, 262, 267, 271, 295, 302, 303, 310, 314, 317, 319, 319, 321,323, 324, 325, 327, 328, 329, 330, 331, 332, 333, 334, or 335, saidpositions being defined with reference to SEQ ID NO: 3, and wherein theparent polypeptide has at least 90% homology with SEQ ID NO: 3; c)optionally substituting one or more further amino acids as defined inb); d) preparing the variant resulting from steps a)-c); e) determiningthe specific activity at least one pH and/or the pH optimum of thevariant; and f) selecting a variant having an increased specificactivity at least one pH in comparison to the parent asparaginasepolypeptide and/or an increased pH optimum in comparison to the parentasparaginase polypeptide, thereby to produce an asparaginase polypeptidevariant.
 15. A method according to claim 14, wherein the parentasparaginase polypeptide has the sequence set out in SEQ ID NO:
 3. 16. Amethod according to claim 14 wherein in step b) at least one amino acidresidue corresponding to any of positions 53, 64, 66, 73, 74, 88, 90,91, 111, 161, 211, 228, 262, 267, 295, 303, 310, 314, 317, 321, 324,330, 332, or 333 is substituted; said positions being defined withreference to SEQ ID NO: 3, and wherein the parent polypeptide has atleast 90% homology with SEQ ID NO:
 3. 17. A method according to claim 16wherein in step b) the substituted amino acid residue corresponds to oneor more of Tyr at position 53; Pro at position 64; Pro at position 66;Lys at position 73; Ala at position 74; Tyr or Pro at position 88; Valat position 90; Glu at position 91; Gly at position 111; Leu at position161; Ser at position 211; His at position 228; Cys or His at position262; Tyr at position 267; Ser at position 295; Ser at position 303; Valat position 310; Asp at position 314; Ile at position 317; Thr atposition 321; Gly at position 324; Ser at position 330; Ala at position332; or Glu at position 333; said positions being defined with referenceto SEQ ID NO:
 3. 18. A composition comprising the variant according toclaim
 1. 19. A process for the production of a food product involving atleast one heating step, comprising adding one or more asparaginaseenzymes according to claim 1 to an intermediate form of said foodproduct in said production process, whereby the enzyme is added prior tosaid heating step in an amount that is effective in reducing the levelof asparaginase that is present in said intermediate form of said foodproduct.